Laboratory Evaluation of Pharyngitis and Pneumonia

1
Laboratory Evaluation of Pharyngitis and
Pneumonia

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

2
Laboratory Evaluation of Respiratory Tract
Infection

• Pathophysiology of pharyngitis, acute pneumonia,
  nosocomial pneumonia, and chronic pneumonia
  prototypes
• Microbiology of respiratory tract infection
• Clinical signs and symptoms of respiratory tract
  infection

3
Laboratory Evaluation of Respiratory Tract
Infection

• Specimen collection, staining, evaluation, and
  culture
• Interpretation of respiratory cultures
• Non-culture evaluation of respiratory tract
  infection

4
Pathophysiology of pharyngitis and pneumonia
prototypes

• Streptococcal pharyngitis
• Acute pneumococcal pneumonia
• Ventilator-associated nosocomial pneumonia
• Tuberculosis (chronic pneumonia)

5
Pathophysiology of Streptococcal Pharyngitis

• Occurs predominantly among children 5-15 years of
  age
• Streptococcus pyogenes spread person-to-person by
  droplets of saliva or nasal secretions
• Pharyngeal carriage of Streptococcus pyogenes
  frequent in asymptomatic individuals (15-20 in
  schoolchildren)
• Interference by viridans streptococci commensals
  likely important in balance between pharyngeal
  colonization and invasive infection by
  Streptococcus pyogenes

6
Pathophysiology of Streptococcal Pharyngitis

• Strain-related virulence factors (hyaluronic acid
  capsule, M protein, lipoteichoic acid) major
  determinants of pharyngeal disease
• Streptococcal tonsillopharyngitis characterized
  by intense neutrophilic inflammatory response
  with edema, erythema, and (often) gray-yellow
  exudation
• Complications acute rheumatic fever, acute
  glomerulonephritis, and invasive infection
  (retropharyngeal or peritonsillar abscess)

7
Pathophysiology of Acute Pneumococcal Pneumonia

• Occurs in older age groups (mid-50s to late
  60s) with chronic underlying disease (COPD,
  alcohol abuse, neurological disease such as
  stroke, congestive heart failure, malignancy,
  liver disease including hepatitis and cirrhosis,
  diabetes mellitus)
• Colonization of nasopharynx by Streptococcus
  pneumoniae frequent in healthy individuals (5-10
  of adults, 20-40 of childen)
• Colonization acquired by extensive and close
  person-to-person contact, invasive disease
  (pneumonia) develops in predisposed hosts
• Predisposing factors impaired cough and
  epiglottic reflexes, disrupted bronchial
  mucociliary clearance, diminished production of
  opsonizing antibody

8
Pathophysiology of Acute Pneumococcal Pneumonia

• With aspiration of Streptococcus pneumoniae into
  deep respiratory tract, bacteria proliferate in
  alveolar spaces
• Alveolar macrophages initial line of defense, but
  if microbial mass exceeds ability of macrophages
  to contain infection, macrophages produce
  interleukin-8 (a potent chemotaxin for
  neutrophils)
• Activation of alternate complement by bacteria
  also provokes intra-alveolar neutrophilic
  exudation and pulmonary consolidation
• Complications empyema (5), bacteremia (25
  preantibiotic era)

9
Pathophysiology of Ventilator-Associated
Pneumonia (VAP)

• Hospital-acquired (nosocomial) pneumonia
  pneumonia that occurs 48 hr or more after
  admission to the hospital, and was not incubating
  at the time of admission
• Healthcare associated pneumonia pneumonia
  associated with 2 or more days of hospitalization
  within previous 90 days, residence in a nursing
  home or long-term care facility, receipt of
  intravenous antibiotic, chemotherapy, or wound
  care within the previous 30 days, or attendance
  at a hospital or hemodialysis clinic
• Ventilator-associated pneumonia pneumonia that
  develops more than 48-72 hr after endotracheal
  intubation

10
Pathophysiology of Ventilator-Associated
Pneumonia (VAP)

• Sources of pathogens include the environment
  (water and equipment) and bacteria transferred
  between patients by staff
• Severity of underlying disease, prior surgery,
  exposure to antibiotics, and use of invasive
  respiratory equipment major risk factors
• Intubation and mechanical ventilation increase
  the risk of hospital-acquired pneumonia 6- to
  21-fold

11
Pathophysiology of Ventilator-Associated
Pneumonia (VAP)

• Aspiration of oropharyngeal pathogens (aerobic
  gram-negative bacilli, Staphylococcus aureus) by
  leakage around the endotracheal tube cuff major
  route of entry for bacteria into lower
  respiratory tract
• Infected biofilm in the endotracheal tube with
  subsequent embolization to distal airways may be
  important
• Complications drug-resistant pneumonia,
  polymicrobial pneumonia, superinfection with
  Pseudomonas aeruginosa or Acinetobacter with high
  mortality, empyema, lung abscess, Clostridium
  difficile colitis, occult infection, bacteremic
  sepsis with multiple organ involvement

12
Pathophysiology of Tuberculosis (Chronic
Pneumonia)

• Source of Mycobacterium tuberculosis an infected
  patient with active pulmonary disease
• M. tuberculosis transmitted by coughing,
  sneezing, or talking with release of infected
  respiratory secretion as aerosols (droplet
  nuclei)
• Droplet nuclei (1-5 µm) penetrate deep alveolar
  spaces and M. tuberculosis infects non-immune
  macrophages as facultative intracellular
  pathogens

13
Pathophysiology of Tuberculosis (Chronic
Pneumonia)

• Active but clinically silent infection is
  contained (but not eliminated) within 2-3 months
  by CD4 T-cell dependent macrophage activation
• Latent infection reactivates due to decrement in
  CD4 T-lymphocyte function (age, AIDS,
  immunosuppressive therapy, anti-TNF-a antibody
  treatment of rheumatoid arthritis, end stage
  renal disease, bronchogenic carcinoma)

14
Pathophysiology of Tuberculosis (Chronic
Pneumonia)

• Type 4 hypersensitivity reaction produces chronic
  mononuclear inflammation of the lung which does
  not resolve in the absence of chemotherapy
• Complications progressive tissue necrosis
  (cavitation), high mortality without specific
  drug therapy

15
Microbiology of Respiratory Tract Infection

• Pharyngitis
• Acute pneumonia
• Ventilator-associated pneumonia
• Chronic pneumonia

16
Microbiology of Pharyngitis (Usual Bacterial
Causes)

• Streptococcus pyogenes (15-30 of cases in
  children, 10 in adults)
• Beta-hemolytic group C and G streptococci
  (associated with foodborn outbreaks of
  pharyngitis)
• Arcanobacterium haemolyticum (exudative
  pharyngitis similar to ß-hemolytic streptococci,
  associated with diffuse erythematous
  maculopapular rash on the extremities and trunk)

17
Microbiology of Pharyngitis(Unusual Bacterial
Causes)

• Neisseria gonorrhoeae (asymptomatic or mild
  pharyngitis)
• Corynebacterium diphtheriae (tonsillar and
  pharyneal inflammatory pseudo- membrane)
• Yersinia enterocolitica (exudative pharyngitis
  associated with ingestion of contaminated food or
  drink)

18
Microbiology of Pharyngitis(Non-Bacterial Causes)

• Epstein-Barr virus (exudative pharyngitis in
  infectious mono- nucleosis)
• Adenovirus types 3, 4, 7, 14, 21 (exudative
  pharyngitis accompanied by conjunctivitis)
• Herpes simplex virus (exudative pharyngitis
  associated with palatal vesicles and shallow
  ulcers)

19
Microbiology of Pharyngitis(Common Cold,
Influenza, and HIV-1)

• Exudative pharyngitis rarely associated with
  common cold and influenza virus
• Common cold viruses rhinoviruses (100 types and
  1 subtype), coronavirus (gt3 types), and
  parainfluenza virus (types 1-4)
• Febrile pharyngitis (hyperemia without exudation)
  characteristic of primary HIV-1 infection after
  3-5 week incubation period, followed in
  approximately 1 week by development of
  lymphadenopathy

20
Microbiology of Acute Community-Acquired Pneumonia

• Streptococcus pneumoniae (16-60 of cases)
• Haemophilus influenza (3-38 of cases)
• Aerobic gram-negative bacteria (7-18 of cases
  with half due to Pseudomonas aeruginosa)
• Staphylococcus aureus (2-5 of cases)
• Legionella species (2-15 of cases)
• Mycoplasma pneumoniae (2-14 of cases)
• Chlamydophila pneumoniae (5-15 of cases)
• Influenza virus (5-12, varies with season)

21
Microbiology of Acute Nosocomial Pneumonia Gram
Negative Bacteria

• Pseudomonas aeruginosa (16 of cases)
• Enterobacter species (11 of cases)
• Klebsiella pneumoniae (7 of cases)
• Other Enterobacteriaceae (9 of cases)
• Acinetobacter species (3 of cases)
• Legionella species (0-2 of cases)
• Haemophilus influenza (0-2 of cases)
• Other gram-negative bacilli (0-10 of cases)
• Total Due to Gram-Negative Bacteria 46-60 of
  Cases
• Carroll, JCM 403115-3120, 2002

22
Microbiology of Acute Nosocomial Pneumonia Gram
Positive Bacteria

• Staphylococcus aureus (17 of cases)
• Streptococcus pneumoniae (2-20 of cases)
• Other (2-5)
• Total Due to Gram-Positive Bacteria 21-42 of
  Cases
• Carroll, JCM 403115-3120, 2002

23
Microbiology of Acute Nosocomial Pneumonia Other
Causes

• Anaerobes (10-20 of cases)
• Fungi (0-10 of cases)
• Mixed (13-54 of cases)
• Total Due to Other Causes 23-84 of cases)
• Carroll, JCM 403115-3120, 2002

24
Microbiology of Chronic Pneumonia Most Common
Causes

• Mycobacterium tuberculosis
• Mycobacteria other than tuberculosis (M.
  kansasii, M. avium complex)
• Endemic dimorphic fungi (Coccidioides immitis,
  Histoplasma capsulatum, Blastomyces dermatiditis)
• Other mycoses (Cryptococcus neoformans,
  Aspergillus species)
• Mixed aerobic and anaerobic bacteria

25
Microbiology of Chronic Pneumonia Infrequent
Causes

• Nocardia species
• Rhodoccus equi
• Burkholderia pseudomallei
• Actinomyces israelii

26
Chronic Pneumonia Syndrome Non-Infectious Causes

• Carcinoma (Primary or Metastatic)
• Lymphoma
• Cystic Fibrosis
• Sarcoidosis
• Amyloidosis
• Pneumoconiosis
• Cryptogenic organizing pneumonia
• Lymphangioleiomyomatosis

27
Clinical Signs and Symptoms of Respiratory Tract
Infection

• Pharyngitis
• Acute pneumonia
• Ventilator-associated pneumonia
• Chronic pneumonia

28
Clinical Signs and Symptoms of Acute Suppurative
Pharyngitis

• Marked pharyngeal pain, painful swallowing
• Fiery red hyperemia of pharyngeal mucosa with
  patchy, gray-yellow exudate on tonsils and uvular
  edema
• Temperature gt39.4oC
• Leukocyte count gt12,000/mm3
• Headache, chills, abdominal pain (variable)

29
Clinical Signs and Symptoms of Acute Community
Acquired Pneumonia

• Sudden onset of chill followed by fever,
  pleuritic chest pain, and cough productive of
  mucopurulent sputum
• Fatigue, anorexia, sweats, and nausea (variable)
• Tachypnea (respiratory rate gt24-30
  breaths/minute), tachycardia (pulse rate gt100
  beats/minute), rales, signs of consolidation
  (variable)
• Leukocyte count 15,000-30,000/mm3
• Thick, purulent, often rust-colored sputum
• Chest X-ray patchy infiltrates, lobar
  consolidation, pleural effusions

30
Clinical Signs and Symptoms of Atypical Pneumonia
Syndrome

• Sore throat and hoarseness initially
• Fever, malaise, coryza, headache, and cough with
  variable sputum production
• Leukocyte gt10,000/mm3 in 20 of cases
• Chest X-ray usually indicates more extensive
  pulmonary involvement than clinical findings
  suggest, with unilateral or bilateral patchy
  infiltrates in a bronchial or peribronchial
  distribution
• Extrapulmonary findings with Legionella
  pneumophila mental status changes, loose stools
  or diarrhea, bradycardia, elevated liver enzymes,
  hypophosphatemia, hyponatremia, elevated serum
  lactate dehydrogenase, and elevated serum
  creatinine levels

31
Clinical Signs and Symptoms of Chronic Pneumonia

• Initially fever, chills, and malaise
• Progressive anorexia and weight loss
• Pulmonary symptoms appear later with worsening
  cough productive of sputum, dyspnea, hemoptysis,
  and/or pleuritic chest pain
• Leukocyte count often normal (exceptions
  pancytopenia in miliary tuberculosis,
  neutrophilic leukocytosis in pulmonary
  actinomycosis)
• X-ray findings nodular or rounded lesions,
  cavities, with characteristic involvement of
  upper lobes (tuberculosis, histoplasmosis)

32
Specimen Collection, Staining, Evaluation, and
Culture

• Pharyngitis throat swab
• Acute pneumonia sputum
• Ventilator-associated pneumonia bronchoalveolar
  lavage (BAL), bronchial brushings
• Chronic pneumonia sputum, BAL

33
Pharyngitis Throat Swab for Streptococcus
pyogenes

• Collect two throat swabs, one for direct antigen
  testing (Lancefield group A antigen) and one for
  culture
• Throat swabs transported in Amies or modified
  Stuarts medium and refrigerated if not tested
  within a few hours of collection
• Test one swab for Lancefield group A antigen, if
  positive (sensitivity 31-95) culture not
  necessary, if negative culture performed
• Inoculate the second swab to sheep blood agar
  (SBA)

34
Pharyngitis Throat Swab for Streptococcus
pyogenes

• Streak SBA plate for isolation and make several
  stabs, incubate for 48 hours at 35oC with air
• After 18-24 hours 0.5 mm translucent or
  transparent colonies with wide zone of
  ß-hemolysis (2-4 times the colony diameter) and
  enhanced ß-hemolysis in stabs, gram-positive
  cocci by Grams stain, negative for catalase
• If PYR positive, confirm as S. pyogenes by
  Lancefield grouping (group A)
• SBA plate culture negative at 24 hours are
  incubated an additional 24 hours
• SBA plate culture the gold standard for
  laboratory confirmation of group A streptococcal
  pharyngitis

35
Pharyngitis Throat Swab for Group C and G
Streptococci and Arcanobacterium haemolyticum

• Colonies identical in appearance to group A
  streptococci on SBA plate, but PYR negative and
  positive by Lancefield grouping for group C or
  group G, report as group C or G ß-hemolytic
  Streptococcus
• After 48-72 hours incubation in SBA plate
  culture, 0.5 mm colonies showing ß-hemolysis,
  irregular (diphtheroid-shaped) gram-positive rods
  by Grams stain, catalase negative, positive for
  reverse CAMP test, report as Arcanobacterium
  haemolyticum
• No direct antigen test for group C or group G
  Streptococcus, or for Arcanobacterium
  haemolyticum

36
Pharyngitis Throat Swab for Unusual Bacteria

• Neisseria gonorrhoeae Inoculate to modified
  Thayer-Martin medium, incubate 72 hours in
  presence of 5 CO2 (gram-negative diplococci by
  Grams stain)
• Corynebacterium diphtheriae Inoculate to
  Loefflers serum medium and potassium tellurite
  agar, incubate 48 hours in 5 CO2 (methylene blue
  stain of irregular gram-positive rods growing on
  Loefflers reveals metachromatic granules,
  colonies on tellurite agar black with brown halo,
  gram-positive coryneform rods by Grams stain)
• Yersinia enterocolitica Inoculate to CIN
  (cefsulodin-irgasan-novobiocin) agar, selective
  for Yersinia which forms red bulls-eye colonies
  due to mannitol fermentation with neutral red pH
  indicator, gram-negative coccobacilli by Grams
  stain)

37
Acute Pneumonia SputumGeneral Principles

• Upper respiratory tract to the larynx major
  source of bacterial contamination of sputum
  specimens
• The tracheobronchial tree sterile or harbors
  sparse numbers of bacteria during good health
• Most bacterial pathogens responsible for lower
  airway infection present at gt106/mL respiratory
  secretions, equivalent to moderate to heavy
  growth on primary isolation plates
• Expectorated sputum should be obtained by deep
  coughing and processed for Grams stain and
  culture within 1 hour or refrigerated

38
Acute Pneumonia SputumGrams Stain for
Assessment of Quality

• Gram-stained smear of sputum screened
  microscopically at low power (X100) magnification
  for numbers of squamous epithelial cells and
  leukocytes
• gt10 squamous epithelial cells/low power field
  indicates gross contamination of specimen by
  oropharyngeal contents (saliva) and should not be
  cultured
• gt25 leukocytes/low power field indicates purulent
  respiratory secretions
• Optimal sputum specimen lt10 squamous epithelial
  cells and gt25 leukocytes/low power field

39
Acute Pneumonia SputumGrams Stain for
Etiological Diagnosis

• Streptococcus pneumoniae Predominance of
  lancet-shaped gram-positive diplococci
• Haemophilus influenzae Small faintly-stained
  gram-negative coccobacilli
• Staphylococcus aureus Gram-positive cocci in
  tetrads and grape-like clusters

40
Acute Pneumonia SputumGrams Stain for
Etiological Diagnosis

• Definitive Presumptive Diagnosis1
• Pneumococcal H.
  influenzae Pneumonia
  Pneumonia
• Sens 57.0 82.3
  
• Spec 97.3 99.2
• PPV 95.1 93.3
• NPV 71.3 97.6
• 1Definitive normally sterile specimen culture
  positive
• Presumptive sputum culture positive
• Roson et al., Clin Inf Dis 31869-874, 2000

41
Acute Pneumonia SputumGrams Stain for
Etiological Diagnosis

• Definitive
  Diagnosis1
• Pneumococcal H.
  influenzae Pneumonia
  Pneumonia
• Sens 35.4 42.8
  
• Spec 96.7 99.4
• PPV 90.6 75.0
• NPV 62.7 98.2
• 1Definitive normally sterile specimen culture
  positive
• Roson et al., Clin Inf Dis 31869-874, 2000

42
Acute Pneumonia Sputum Culture

• Inoculate sputum specimens acceptable by Grams
  stain screen to sheep blood, chocolate, and
  MacConkey agar
• Streak for isolation
• Incubate at 35oC in 3-5 CO2 for 2 days
• Report potential respiratory pathogens with
  predominant growth on sheep blood agar (gt10
  colonies in primary streak, gt5 colonies in
  secondary streak, and any growth in tertiary
  streak)

43
Potential Respiratory PathogensHospital Acquired
Infection1

• Enterobacteriaceae
• Staphylococcus aureus
• Pseudomonas aeruginosa
• Other nonfermentative gram-negative bacilli
• Less commonly, same as community acquired
• 1Cultivable by routine sputum culture procedures
• ASM Manual 2003

44
Potential Respiratory PathogensCommunity
Acquired Infection1

• Streptococcus pneumoniae
• Haemophilus influenzae
• Moraxella catarrhalis
• Nocardia asteroides complex
• Pasteurella multocida
• 1Cultivable by routine sputum culture procedures
• ASM Manual 2003

45
Oropharyngeal Commensals1

• Viridans group streptococci other than
  Streptococcus pneumoniae
• Coagulase-negative Staphylococcus
• Neisseria (saprophytic species)
• Corynebacterium species
• 1Cultivable by routine sputum culture but not
  reported

46
Acute Pneumonia Sputum Culture

• Using quantitation of streaked blood agar,
  predominant growth of one to three potential
  respiratory tract pathogens generally observed
• Sensitivity of sputum culture for bacteremic
  pneumococcal pneumonia 50-55 (equivalent to a
  coin flip)
• Sensitivity of sputum culture for bacteremic
  Haemophilus influenzae pneumonia 53-66
• Contamination of sputum culture by colonizing
  gram-negative bacilli 32 of sputum cultures
• Pleural effusion and blood cultures from an
  uncontaminated specimen source (relatively) and
  thus highly specific with recovery of potential
  respiratory pathogens, including anaerobic
  bacteria

47
Acute Pneumonia Etiological Organisms
Non-Cultivable by Routine Sputum Cultures

• Legionella (culture on buffered charcoal yeast
  extract medium, urine antigen, serology)
• Chlamydophila pneumoniae (serology)
• Mycoplasma pneumoniae (serology)
• Anaerobic bacteria (protected bronchoscope
  brushing for anaerobic culture)

48
Ventilator-Associated Pneumonia

• Bronchoalveolar lavage fluid. Fiber-optic
  bronchoscope wedged tightly into bronchial
  orifice of involved segment, and distal airspaces
  lavaged with a minimum of 140 ml of fluid
  (approximately 100 million alveoli are sampled)
• Protected specimen brush. Protected
  brush-catheter consists of a double-lumen
  catheter with a distal occluding plug inserted
  through the inner suction channel of a
  bronchoscope wedged into the bronchial orifice of
  an involved area, brushings obtained, the brush
  severed from a retracting wire, and the brush
  placed directly into 1 ml of sterile saline.

49
Bronchoalveolar Lavage Fluid

• Grams stain presence of intracellular bacteria
  indicative of potential pathogen presence of gt1
  of all cells as squamous epithelial cells
  indicates falsely elevated counts of potential
  pathogens due to oropharyngeal contamination
• Quantitative cultures obtained by inoculation of
  sheep blood agar with 0.01 and 0.001 ml aliquot
  of bronchoalveolar lavage fluid
• MacConkey and chocolate agars also inoculate to
  enhance recovery of gram-negative bacteria
  (including fastidious organisms) with
  polymicrobial infections

50
Bronchoalveolar Lavage Fluid

• Recovery of lt10,000 bacteria/ml of lavage fluid
  suggests contamination
• Recovery of gt100,000 potential respiratory
  pathogen/ml indicates infection
• Detection of 10,000-100,000 potential respiratory
  pathogen/ml a gray zone (prior antibiotic
  therapy, inadvertent contamination of lavage
  fluid)

51
Protected Specimen Brush

• Recovery of gt1,000 of potential respiratory
  pathogen/ml saline indicates infection

52
Chronic PneumoniaSputum

• Grams Stain for Nocardia
• Acid fast stain for mycobacteria
• Gomori methenamine silver or periodic acid-Schiff
  stain for fungi
• Cytologic preparations for neoplastic cells and
  fungi

53
Predictive Power of Acid-Fast Smear for
Tuberculosis

• Sputum Volume
• Any Volume Minimum of 5
  ml
• Sens 72.5 92.0
  
• Spec 98.2 98.5
• PPV 56.3 79.9
• NPV 99.1 99.5
• 1Values of sensitivity, specificity, PPV, and NPV
  calculated using culture results positive or
  negative for Mycobacterium tuberculosis
• Warren et al. Am J Resp Crit Care Med
  1611559-1562, 2000

54
Chronic PneumoniaSputum Culture

• Mycobacteria Lowenstein-Jensen slants,
  Middlebrook agars (7H10, 7H11, S7H11),
  Middlebrook broths
• Fungi Brain heart infusion (BHI) agar,
  inhibitory mold agar (contains chloramphenicol),
  cornmeal agar

55
Recommended Readings

• Bisno, AL. Pharyngitis, in Mandell, Douglas, and
  Bennets Principles and Practice of Infectious
  Diseases, 6 ed., 2005, pp. 752-758.
• Donowitz, GR, Mandell, GL. Acute pneumonia.
  Ibid., pp. 819-845.
• Pappas, PG, Dismukes, WE. Chronic pneumonia.
  Ibid., pp. 857-869.

56
Recommended Readings

• Roson et al. Prospective study of the usefulness
  of sputum gram stain in the initial approach to
  community-acquired pneumonia requiring
  hospitalization. Clin Inf Dis 200031869-874.
• Bartlett et al. Laboratory diagnosis of lower
  respiratory tract infections. Cumitech 7A,
  American Society for Microbiology, September
  1987.
• Carroll, KC. Laboratory diagnosis of lower
  respiratory tract infections controversy and
  conundrums. J Clin Micro 2002403115-3120.

57
Recommended Readings

• Official statement of the American Thoracic
  Society and the Infectious Diseases Society of
  America. Guidelines for the management of adults
  with hospital-acquired, ventilator-associated,
  and healthcare-associated pneumonia. Am J Respir
  Crit Care Med 2005171388-416.
• Warren et al. A minimum 5.0 ml of sputum
  improves the sensitivity of acid-fast smear for
  Mycobacterium tuberculosis. Am J Respir Crit
  Care Med 20001611559-1562.

58
Recommended Readings

• Thomson,RB, Jr, Miller, JM. Specimen collection,
  transport, and processing bacteriology, in
  Manual of Clinical Microbiology, 8th edition,
  2003, pp. 286-330.